MHC-presented peptide vaccines are one of the most promising experimental therapies for melanoma. However, MHC class I peptides that prime CTLs lack dramatic anti-tumor effect in clinical trials. Co-immunization with MHC class II peptides substantially enhances CTL potency, augments antibody development and provides memory. A drawback to this approach is that MHC class II epitope bind poorly, therefore they are weak immunogens. Antigen Express scientists found that coupling the li-Key segment of the immunoregulatory li-protein to the N-terminus of a MHC class II epitope enhances the potency of class II epitope peptide presentation 200 times more than the epitope-only peptide. Our studies in immunized mice demonstrate 4-6 fold enhancement of Th1 cells by with li-Key/MHC II hybrid as measured by ELISPOT. In vitro li-Key/HER-21/neu(MHC II epitope) hybrids potently stimulate peripheral blood or draining lymph node lymphocytes from breast cancer patients. Applying these methods to melanoma, we will define the most potent HLA-DR-restricted li-Key/tyrosinase(MHC II) and li-key/gpl00(MHC II) hybrids for a clinical trial by our collaborator, Dr. Jedd Wolchok of Memorial Sloan Kettering Cancer Center (MSKCC). He will use these MHC class II hybrids to potentiate CTL and clinical responses to previously used tyrosinase and gp100 HLA-A2-restricted CTL epitope peptide vaccines. Here we will define the optimal sequence of MHC class II epitopes for incorporation in new hybrids, optimal spacer length, and dose/dosage and immune response parameters in mice. In parallel with studies using human cells in vitro, Dr. MigueI-Angel Perales from MSKCC will confirm our structure-activity findings. Toxicology studies in mice and rabbits of one GMP batch of the optimal peptide(s) will complete our preclinical studies of this SBIR, supporting a clinical trial at Memorial Sloan-Kettering.